G protein-coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from the sensation of light to the contractility of the heart. A family of GPCR kinases (GRKs) modulates the activity of these GPCRs by phosphorylating sites in their cytoplasmic loops and C-terminal tails. Although GRKs allow cells to adapt and can protect them from damage incurred by sustained signaling, aberrant GRK activity has been associated with human disease such as hypertension and heart failure. Inhibition of GRK activity is also expected to enhance the action of the many drugs that target GPCRs. In the last five years, our lab has made significant progress in understanding the structure and function of this kinase family. We have produced high resolution crystal structures that represent all three GRK subfamilies, including that of GRK1 (rhodopsin kinase), GRK2 (2-adrenergic receptor kinase 1), and GRK6, as well as structures of GRK2 in complex with heterotrimeric G1q and G23 subunits. While much has been learned about the modular structure of GRKs, their interactions with G proteins, and their configuration at the membrane, only recently have we determined a crystal structure that permits us to rationally test how GRKs recognize and are allosterically activated by GPCRs. In the first aim of this proposal, we test hypotheses derived from our breakthrough structure of GRK6 in a closed conformation, wherein a conserved N-terminal helix docks with the kinase domain and stabilizes it in a more active state. This helix extends from the kinase domain such that it could interact with a GPCR in a manner analogous to how the C-terminal helix of transducin binds opsin. The second aim is devoted to crystallographic analysis of GRK-receptor complexes. We will pursue structures of the closed conformation of GRK6 in complex with substrate peptides derived from the phosphoacceptor sites of GPCRs. To help define how GRKs dock on the receptor, we will develop peptides and/or peptidomimetics derived from the N-terminal helices of GRKs that bind with high affinity to activated bovine or cephalopod rhodopsin for co-crystallization screens. We will also attempt to determine structures of these prototypical GPCRs in complex with full-length GRKs that we engineer to more readily assume a closed conformation. Our final aim is to use a crystallographic approach to define the molecular basis for how a novel RNA aptamer inhibits GRK2 with high affinity and selectivity. We will develop an assay to screen for selective compounds that target key pockets on the surface of GRK2 bound by the aptamer, and will attempt to engineer new aptamers that are selective for GRK6. Understanding how GPCRs activate GRKs and characterizing the unique and functionally critical sites on these enzymes is key to the development of agents that can selectively regulate GRK function in cells. PUBLIC HEALTH RELEVANCE: G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and thereby regulate the activity of most of the ~800 GPCRs in the human genome. Some GRKs, such as GRK2, are strongly implicated in the progression of cardiovascular disease and hypertension. This proposal investigates the molecular basis for how GRKs recognize and are regulated by their target GPCRs, and seeks to structurally characterize a novel GRK inhibitor that could lead to the development of therapeutic agents or new molecular tools to dissect GRK function in cells.